PROJECT SUMMARY The HIV epidemic in Central Asia, including Kazakhstan was originally confined largely to persons who inject drugs (PWID), but is now spreading through sexual- and migrant-associated transmissions. Central Asia represents the world's most rapidly growing HIV epidemic region. Phylogenetic transmission clusters, composed of genetically similar HIV sequences (<1.5% diversity), represent recently-acquired/rapidly- transmitting infections (transmission ?hotspots?). Our objective is to identify PWID-associated currently at-risk populations, sothey may be linked to services expeditiously.Our specific aims are: 1. Obtain sequences from well-characterized Kazakhstani HIV-infected blood samples from PWID and other high risk persons for phylodynamic and statistical modeling of HIV transmission clusters: To achieve this we will: a) Analyze previously generated 800 HIV pol sequences, and prospectively collect 400 blood samples, from consenting HIV-positive PWID and other high-risk groups, including men who have sex with men (MSM), heterosexuals, and male/female sexual partners; b) Administer a validated questionnaire to 400 consenting participants to characterize gender, sexual/social contacts, travel history, and risk behaviors; c) Include the same data for the above-mentioned ?800 HIV historic samples collected and previously characterized by our collaborators; d) Extract HIV RNA/DNA from the prospective blood samples to carry out amplification and sequencing of the HIV pol gene. 2. We will use these HIV DNA sequences and linked questionnaire data to identify PWID-associated HIV transmission clusters and common clinical, sociodemographic and behavioral characteristics to focus resources towards the most relevant settings/high-risk behaviors. These clusters will be elucidated by: a) Using phylogenetic analysis to identify HIV subtypes/recombinant forms; b) Determining profiles of drug resistance and trends of its spatiotemporal transmission; c) Using genetic distance to the closest sequence as a measure of clustering and questionnaire data to identify subpopulations exhibiting rapid transmission; d) Using our prospectively generated 400 HIV sequences along with historic 800 Kazakhstani (and central Asian sequences obtained from open-access databases) to determine whether identified transmission clusters are newly emerging and/or rapidly expanding, and e) Inferring intra-Kazakhstan and Central Asian movements of HIV-1 by applying structured coalescent phylogenetic models. These analyses will be cross-confirmed using the open-source near real-time tracking platform, nextHIV, that performs automated alignment, subtyping, genotypic resistance determination, phylogenetic reconstruction and genetic distance clustering. The ultimate goal of our R03 small grant is to provide evidence regarding transmission hotspots that will help target emerging/expanding transmission clusters, stimulating both HIV control and prevention, and HIV molecular virology research in Kazakhstan and elsewhere in Central Asia.